Vertebrate telomerase genes and proteins and uses thereof

Abstract

Nucleic acid molecules encoding vertebrate telomerase are provided. Gene products, expression vectors and host cells suitable for expressing telomerase are also provided. Methods for identifying inhibitors of telomerase activity and inhibitor compositions are disclosed.

1. An isolated nucleic acid molecule encoding a splice variant of a gene sequence capable of being spliced to result in a reference human telomerase encoding SEQ ID No: 2, wherein the splice variant has at least one of the following insertions or deletions:
 (a) an insertion of sequences X (comprising SEQ ID No: 32) at nucleotide 1766 of SEQ ID No: 1;  
 (b) an insertion of nucleic acid sequence encoding sequence 1 (SEQ ID No: 24) at nucleotide 1950 of SEQ ID No: 1;  
 (c) a deletion of nucleotides 2131 through 2166 of SEQ ID No: 1;  
 (d) a deletion of nucleotides 2287 through 2468 of SEQ ID No: 1;  
 (e) an insertion of sequence 2 comprising SEQ ID No: 29 at nucleotide 2843 of SEQ ID No: 1; and  
 (f) an insertion of nucleic acid sequence encoding sequence 3 (SEQ ID No: 31) at nucleotide 3157 of SEQ ID No: 1,  
 and wherein the splice variant does not encode SEQ ID No: 2.  
 
     
     
       2. The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule encodes one of SEQ ID Nos: 35, 37, 39, 42, 44, 46, 48, 50, 52-54, 56-58, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86. 
     
     
       3. An isolated nucleic acid molecule encoding any of SEQ ID Nos: 35, 37, 39, 42, 44, 46, 48, 50, 56-58, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86 or a variant thereof,
 wherein said variant has at least 95% amino acid identity with said amino acid sequences and binds telomerase RNA (hTR) or has telomerase activity; and  
 wherein the nucleic acid does not encode SEQ ID No: 2.  
 
     
     
       4. An isolated nucleic acid molecule consisting of any of SEQ ID Nos: 23, 25, 27, 29, 30, 32, 33, or the complement thereof. 
     
     
       5. An isolated nucleic acid molecule encoding any of SEQ ID Nos: 24, 26, 28, or 31. 
     
     
       6. An expression vector, comprising a heterologous promoter operably linked to a nucleic acid molecule according to any of claims  1 ,  2 - 4 , and  5 . 
     
     
       7. The expression vector of  claim 6 , wherein the vector is selected from the group consisting of bacterial vectors, retroviral vectors, adenoviral vectors and yeast vectors. 
     
     
       8. A host cell containing a vector according to  claim 6 . 
     
     
       9. The host cell of  claim 8 , wherein the cell is selected from the group consisting of human cell, monkey cell, mouse cell, rat cell, yeast cell and bacterial cell. 
     
     
       10. The host cell of  claim 8 , wherein the cell is a human cell. 
     
     
       11. A nucleic acid probe that is capable of specifically hybridizing to a nucleic acid molecule encoding a splice variant of human telomerase according to  claim 1 
 wherein the probe consists of one of SEQ ID Nos: 23, 25, 27, 29, 30, 32 or 33 or the complement thereof;  
 or a fragment of SEQ ID Nos: 23, 29, 30, 31 or 32 or the complement thereof.  
 
     
     
       12. The probe of  claim 11 , wherein the fragment is from 12 to 200 nucleotides long. 
     
     
       13. The probe of  claim 11 , wherein the fragment is from 20 to 50 nucleotides long. 
     
     
       14. The probe of  claim 11 , wherein the nucleic acid molecule is labeled. 
     
     
       15. An isolated nucleic acid molecule consisting of a sequence selected from the group consisting of region 1 (SEQ ID No:23), region α (SEQ ID No:25), region β (SEQ ID No:27), region 2 (SEQ ID No:29), and region 3 (SEQ ID No:30). 
     
     
       16. The nucleic acid molecule of  claim 1 , wherein the splice variant of human telomerase has at least a deletion of nucleotides 2131-2166 of SEQ ID No: 1. 
     
     
       17. The complement of the nucleic acid molecules of any of claims  1 ,  2 ,  3 ,  4  and  5 . 
     
     
       18. The nucleic acid molecule of any of claims  2 ,  3 ,  4  and  5 , wherein said molecule is a DNA molecule. 
     
     
       19. The nucleic acid molecule of any of claims  1 ,  2 ,  3 ,  4  and  5 , wherein said molecule is an RNA or cDNA molecule. 
     
     
       20. A pair of oligonucleotide primers that amplify nucleic acid sequence of human telomerase containing a splice junction, wherein only one primer of each primer pair flanks nucleotide 1950, 2131-2166, 2287-2468, 2843, or 3157 as presented in  FIG. 1  (SEQ ID No: 1) and the other primer of the pair has sequence corresponding to all or a portion of one of the sequences presented in  FIG. 10  (SEQ ID Nos: 23, 25, 27, 29, 30, 32, 33) or complements thereof. 
     
     
       21. The probe of  claim 14 , wherein the label is a chemiluminescent label, a radioactive label, or biotin. 
     
     
       22. The nucleic acid molecule of  claim 1 , wherein the splice variant of human telomerase has at least an insertion of nucleic acid sequence encoding sequence 3 (SEQ ID No:31) at nucleotide 3157 of SEQ ID No: 1. 
     
     
       23. An oligonucleotide consisting of 15-100 contiguous nucleotides of one of the sequences selected from the group consisting of SEQ ID Nos. 23, 29, 30, 32, 33 or complements thereof. 
     
     
       24. The oligonucleotide of  claim 23 , wherein the oligonucleotide is labeled. 
     
     
       25. The oligonucleotide of  claim 24 , wherein the label is a radiolabel, a chemiluminescent label, or biotin. 
     
     
       26. A pair of nucleotide primers that amplify sequence selected from the group consisting of region 1 (SEQ ID No: 23), region α (SEQ ID No: 25), region β (SEQ ID No: 27), region 2 (SEQ ID No: 29), region 3 (SEQ ID No: 30), region X (SEQ ID No: 32), wherein the primers comprise at least 15 contiguous nucleotides of one of SEQ ID Nos: 23, 25, 27, 29, 30, 32, or 18 or complements thereof and wherein the primers are from 15 to 50 nucleotides in length. 
     
     
       27. A method of determining a pattern of telomerase RNA expression in cells, comprising:
 (a) preparing cDNA from mRNA isolated from the cells,  
 (b) amplifying the cDNA using primers that amplify a splice variant of nucleic acid encoding human telomerase to form an amplified product and  
 (c) hybridizing the amplified product with one or more of the following:  
 all or at least 15 contiguous nucleotides of the sequence of region 1 (SEQ ID No: 23), all or at least 15 contiguous nucleotides of the sequence of region β (SEQ ID No: 27), all or at least 15 contiguous nucleotides of the sequence of region 2 (SEQ ID No.: 29), all or at least 15 contiguous nucleotides of the sequence of region 3 (SEQ ID No: 30), or all or at least 15 contiguous nucleotides of the sequence of region X (SEQ ID No: 32); or a complement thereof; and  
 (d) detecting hybridization;  
 therefrom determining the pattern of telomerase RNA expression.  
 
     
     
       28. A method of determining a pattern of telomerase RNA expression in cells, comprising,
 (a) preparing cDNA from mRNA isolated from the cells,  
 (b) amplifying the cDNA using primers that amplify a splice variant of nucleic acid encoding human telomerase to form an amplified product and  
 (c) hybridizing the amplified product with two or more of the following:  
 all or at least 15 contiguous nucleotides of the sequence of region 1 (SEQ ID No: 23), all or at least 15 contiguous nucleotides of the sequence of region α (SEQ ID No: 25), all or at least 15 contiguous nucleotides of the sequence of region β (SEQ ID No: 27), all or at least 15 contiguous nucleotides of the sequence of region 2 (SEQ ID No: 29), all or at least 15 contiguous nucleotides of the sequence of region 3 (SEQ ID No: 30), all or at least 15 contiguous nucleotides of the sequence of region X (SEQ ID No: 32) or all or at least 15 contiguous nucleotides of the sequence of region Y (SEQ ID No: 18); or a complement thereof; and  
 (d) detecting hybridization;  
 therefrom determining the pattern of telomerase RNA expression.  
 
     
     
       29. The method of either of claims  27  or  28 , wherein the contiguous nucleotides contain a label. 
     
     
       30. The method of  claim 29 , wherein the label is radioactive, chemiluminescent, or biotin.